Fetal polypeptides from human liver

ABSTRACT

Novel fetal genes (fls353 and fls485) have been successfully isolated from human fetal liver-derived cDNAs. These genes were specifically expressed in tissues including fetal tissues which are thought to contain a large number of undifferentiated cells and actively differentiating/proliferating cells. High levels of expression of these genes were observed also in a variety of cancer cells. The proteins and genes encoding the proteins can be used as the tool for developing drugs for the treatment of tumors.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 11/800,888, filed on May 8, 2007, now U.S. Pat. No. 7,919,604which is a divisional application of U.S. patent application Ser. No.11/182,592 (now U.S. Pat. No. 7,214,774), filed on Jul. 14, 2005, whichis a divisional application of U.S. patent application Ser. No.09/974,143 (now U.S. Pat. No. 7,022,474), filed on Oct. 9, 2001, whichis a continuation-in-part of PCT/JP00/02281, filed Apr. 7, 2000, whichclaims priority from Japanese patent application Ser. No. 11/103,356,filed Apr. 9, 1999, the disclosures of which are incorporated byreference herein in their entireties.

TECHNICAL FIELD

The present invention relates to proteins encoded by fetal genes theexpression of which is activated in fetal tissues and tumor cells.Proteins of this invention can be utilized as target molecules fordeveloping medicines for the treatment of cancer.

BACKGROUND

Fetal tissues are comprised of many undifferentiated cells thatproliferate actively, highly activated cells, nascent vascularendothelial cells, and so on. Although these are stringently regulatedin fetal tissues and inhibited as individuals mature, this can beconsidered very similar to the state of a solid tumor except that theactivity is regulated. Therefore, some of the genes expressedspecifically in fetal tissues (fetal genes) can be genes involved in thephenomena characteristic of solid tumors such as abnormal growth,immortalization, infiltration, metastasis, and angiogenesis. Inaddition, some diseases other than cancers are also supposed to arisebecause fetal genes, which are repressed in a normal living body, areabnormally activated. Therefore, genes involved in various diseases suchas cancers can be screened by isolating and analyzing fetal genes.Furthermore, development of medicines using novel action mechanisms isthought to be possible by designing drugs targeted on the genes.

Recently, as fetal genes which are assumed to be involved in malignanttransformation, genes for survivin (Nat. Med., 3:917-921, 1997), aurorakinase (EMBO J., 17:3052-3065, 1998) and LYAR (Genes Dev., 7:735-748,1993) have been reported. Their expressions are all activated in coloncancer, leukemia cells, and such, and these genes are thought tocontribution importantly to the malignant transformation. In fact, ithas been demonstrated that survivin has the apoptosis inhibitoractivity, and aurora kinase participates in the cell cycle regulation astheir physiological functions, indicating that acquiring function ofeither of them works favorably for cancer cells.

SUMMARY

The present invention provides novel proteins specifically or morestrongly expressed in fetal tissues, genes encoding the proteins, andpreparation and uses of them.

As a method for isolating a gene involved in the malignanttransformation, many strategies have been hitherto tried where cancercells and normal cells are directly compared so as to identify a commongene the expression level of which is significantly different. However,such a method had a problem that a large number of genes not directlyinvolved in malignant transformation might be isolated as the noise dueto irregularity in the gene expression control, which is one ofcharacteristics of cancer cells, leading to no isolation of importantgenes. Therefore, the present inventors have planned a strategy wheregenes, the expression of which is physiologically controlled fromphysiological necessity, are first selected and examined whether thegenes are abnormally reactivated under non-physiological conditions suchas cancer. Based on this strategy, the inventors have focused on thegenes specifically expressed in fetal tissues as the genes theexpression of which is physiologically controlled from the physiologicalnecessity, and performed their isolations and analyses.

Specifically, first, by the suppression subtractive hybridizationmethod, the present inventors performed the subtraction method with cDNAderived from fetal liver as the tester and cDNA derived from adult liveras the driver to search for fetal genes expressed specifically or morestrongly in the fetal liver.

As a result, they isolated a plurality of genes. Then, they examinedthese genes specifically expressed in fetal tissues for their expressionlevels in cancer cells. As a result, they have succeeded in identifyingnovel genes, “fls353” and “fls485” the expressions of which areactivated in cancer cells such as colon cancer and hepatoma and so on.Cloning of these full-length cDNAs followed by their sequencings haverevealed that, although amino acid sequences of proteins encoded bythese genes comprise several characteristic domain structures, noprotein having a particularly significant homology to these geneproducts could be detected in the database. As a result of furtherstudies on the tissue specificities of these gene expressions in moredetail, the present inventors have found that these genes are expressedin a variety of cancer cells, and also specifically in normal tissues,such as fetal tissues and other tissues which are thought to containmany undifferentiated cells and actively differentiating/proliferatingcells.

These facts have strongly suggested participation of the novel fetalgenes “fls353” and “fls485” in the malignant transformation of cells.Therefore, not only expression inhibitors of these fetal genes areexpected to be used as the anticancer agent but also fetal genes andproteins encoded by the genes according to this invention can be used asthe tool for developing drugs for the treatment of cancer.

The present invention relates to the novel fetal genes “fls353” and“fls485” which are presumed to be involved in the malignanttransformation, and the proteins encoded by these genes as well as theirpreparations and uses, and more specifically, this invention is toprovide the following:

(1) A DNA of any one of (a) to (d) below:

-   -   (a) a DNA encoding a protein comprising the amino acid sequence        of any one of SEQ ID NOs:2, 4 and 6,    -   (b) a DNA comprising a coding region in the nucleotide sequence        of any one of SEQ ID NOs:1, 3 and 5,    -   (c) a DNA encoding a protein that comprises the amino acid        sequence of any one of SEQ ID NOs:2, 4 and 6 in which one or        more amino acids are replaced, deleted, inserted and/or added        and that is functionally equivalent to the protein comprising        the amino acid sequence of any one of SEQ ID NOs:2, 4 and 6, and    -   (d) a DNA that hybridizes under stringent conditions with the        DNA comprising the nucleotide sequence of any one of SEQ ID        NOs:1, 3 and 5 and that codes a protein functionally equivalent        to the protein comprising the amino acid sequence of any one of        SEQ ID NOs:2, 4 and 6.

(2) A DNA encoding a partial peptide of a protein comprising the aminoacid sequence of any one of SEQ ID NOs:2, 4 and 6.

(3) A vector into which the DNA of (1) or (2) is inserted.

(4) A transformant harboring the DNA of (1) or (2) or the vector of (3).

(5) A protein or a peptide encoded by the DNA of (1) or (2).

(6) A method for producing the protein or the peptide of (5), the methodcomprising the steps of culturing the transformant of (4) and recoveringa protein expressed from the transformant or the culture supernatantthereof.

(7) An antibody against the protein of (5).

(8) A polynucleotide that hybridizes with the DNA comprising thenucleotide sequence of any one of SEQ ID NOs:1, 3 and 5 or thecomplementary strand thereof and that comprises at least 15 nucleotides.

(9) A method for screening a compound binding to the protein of (5), themethod comprising the steps of:

-   -   (a) contacting a test sample with the protein or a partial        peptide thereof,    -   (b) detecting a binding activity of the test sample to the        protein or the partial peptide thereof, and    -   (c) selecting a compound comprising the biding activity to the        protein or the partial peptide thereof.

(10) A compound biding to the protein of (5), wherein the compound canbe isolated by the method of (9).

(11) A method for screening a compound that suppresses or promotesexpression of the DNA of (1), wherein the method comprises the steps of:

-   -   (a) contacting a test sample with cells expressing the DNA,    -   (b) detecting the expression of the DNA in the cells, and    -   (c) selecting a compound that decreases or increases the        expression of the DNA compared with that in the case where the        test sample is not contacted with the cells (control).

(12) A method for screening a compound that suppresses or promotesexpression of the DNA of (1), wherein the method comprises the steps of:

-   -   (a) providing cells into which a vector comprising a reporter        gene functionally linked downstream of the expression control        region of the DNA of (1),    -   (b) contacting a test sample with the cells,    -   (c) detecting the activity of the reporter gene in the cells,        and    -   (d) selecting a compound that decreases or increases the        activity compared with that in the case where the test sample is        not contacted with the cells (control).

(13) A compound that suppresses or promotes expression of the DNA of(1), wherein the compound can be isolated by the method of (11) or (12).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph showing the results of the fls353 expressionexamined by northern analysis.

FIG. 2 is a photograph showing the results of the fls353 expression in aclinical sample of E. coli, analyzed by RT-PCR, continued to FIG. 3.

FIG. 3 is a photograph of the continuation of FIG. 2.

FIG. 4 shows the nucleotide sequence of fls353 (SEQ ID NO:1) and itsputative amino acid sequence (SEQ ID NO:2). Amino acids corresponding tothe consensus sequence at the ATP/GTP binding site are shown enclosed bya solid line. Continued to FIG. 5.

FIG. 5 is the continuation of FIG. 4.

FIG. 6 is a photograph showing the results of the fls485 expressionexamined by northern blot analysis.

FIG. 7 is a photograph showing the results of RT-PCR analysis of theexpression of fls485 and a fetoprotein in various cultured cells.

FIG. 8 shows the nucleotide sequence of fls485 L (SEQ ID NO:3) and itsputative amino acid sequence (SEQ ID NO:4). Fls485 S (SEQ ID NO: 5 and6) has the same sequence as fls485 L except that in fls485 S, thenucleotides underlined in FIG. 8 do not exist, and the encircledmethionine (M) is assumed to be used as the initiation codon. Thenucleotide and amino acid sequences of fls485 S are shown in SEQ ID NOs:5 and 6, respectively. Sequence surrounded by the solid square is theamino acid sequence coinciding with the sequence comprisingCys-Xaa-Xaa-Cys-Xaa-Gly-Xaa-Gly (SEQ ID NO: 33), and that surrounded bythe dotted square is the amino acid sequence coinciding with thesequence comprising Cys-Xaa-Xaa-Cys-Xaa-Gly (SEQ ID NO: 34).

FIG. 9 is a photograph showing the results of RT-PCR using a set ofprimers sandwiching the entire coding region of fls485 with cDNA derivedfrom the human fetal liver as the template. The shaded box in the abovefigure represents the coding region of fls485.

FIG. 10 is a photograph showing the results of northern blot analysis ofthe expressions of fls353, fls485 and a fetoprotein in a clinicalhepatoma sample.

FIG. 11 is a photograph showing the results of northern analysis of theexpressions of fls353 and fls485 in a clinical lung cancer sample.

DETAILED DESCRIPTION

This invention relates to the novel proteins “fls353” and “fls485” theexpressions of which are activated in fetal tissues and tumor cells.Nucleotide sequence of cDNA for the novel human fetal gene “fls353” isset forth in SEQ ID NO:1, and two types of nucleotide sequences of cDNAsfor the novel human fetal gene “fls485” which are included in thisinvention are set forth in SEQ ID NO:3 (fls485 L) and 5 (fls485 S),respectively. Amino acid sequences encoded by these cDNAs are set forthin SEQ ID NOs:2 (fls353), 4 (fls485 L) and 6 (fls485 S), respectively.

In normal tissues, those genes have been found to be specificallyexpressed in undifferentiated cells such as in fetal tissues, and intissues which are thought to contain many actively differentiating andproliferating cells. Examination of their expressions in cancer cellsrevealed that they were activated in those such as colon cancer andhepatoma and so on.

As to fetal genes, genes for survivin, aurora kinase and LYAR have beenassumed to be involved in malignant transformation, and it has beenreported that expressions of the genes show a common pattern that theirexpressions in normal tissues are prominent in fetal tissues, especiallyin the fetal liver, and that in adult tissues their expressions areabundant in tissues such as testis and thymus containing many activelyproliferating undifferentiated cells but not detected in other tissuesthan those at all (Nat. Med., 3:917-921, 1997; EMBO J., 17: 3052-3065,1998; and Genes Dev., 7:735-748, 1993). This common expression patternis shared by nearly all of fetal genes in this invention, indicatingthat such a common feature may be produced from the necessity for cellproliferation.

Although mechanisms by which cancer cells reactivate such genesfavorably acting for their own proliferation have not been elucidated,it has been demonstrated in the case of aurora kinase that the geneduplication occurs in high frequency in cancer cells, and thus, this isexpected to be an important cause of its activated expression. Fromthese facts, fetal genes of this invention are thought to be involved inthe malignant transformation of cells mediated by their activation dueto the gene duplication or methylation of DNAs and so on.

Therefore, fetal genes and proteins encoded by the genes of thisinvention can be preferably used not only as the tool for purifying andcloning factors to control the cellular differentiation andproliferation but also as the target for screening candidate compoundsof therapeutics and preventives for disorders such as tumors. Inaddition, the “fls353” and/or “fls485” genes can be applied to themedical treatment such as gene therapy in various cancers.

The term “substantially pure” as used herein in reference to a givenpolypeptide means that the polypeptide is substantially free from otherbiological macromolecules. For example, the substantially purepolypeptide is at least 75%, 80, 85, 95, or 99% pure by dry weight.Purity can be measured by any appropriate standard method known in theart, for example, by column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis.

Accordingly, the invention includes a polypeptide having a sequenceshown as SEQ ID NOs:2, 4 or 6. The invention also includes apolypeptide, or fragment thereof, that differs from the correspondingsequence shown as SEQ ID NOs:2, 4 or 6. The differences are, preferably,differences or changes at a non-essential residue or a conservativesubstitution. In one embodiment, the polypeptide includes an amino acidsequence at least about 60% identical to a sequence shown as SEQ IDNOs:2, 4 or 6, or a fragment thereof. Preferably, the polypeptide is atleast 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more identical toSEQ ID NOs:2, 4 or 6 and has at least one cancer related or fetalfunction or activity described herein. Preferred polypeptide fragmentsof the invention are at least 10%, preferably at least 20%, 30%, 40%,50%, 60%, 70%, or more, of the length of the sequence shown as SEQ IDNOs:2, 4 or 6 and have at least one cancer related or fetal activitydescribed herein. Or alternatively, the fragment can be merely animmunogenic fragment.

This invention also includes proteins functionally equivalent to thehuman “fls353” or “fls485” proteins. Such proteins comprise, forexample, homologous proteins from other organisms corresponding to thehuman “fls353” or “fls485” protein, and mutants of the human “fls353” or“fls485” protein.

In this invention, “functionally equivalent” means that a subjectprotein has functions related to cancer. Whether a protein has functionsrelated to cancer or not can be characterized by, for example, itsprominent expression specificity in cancer cell lines, fetal tissues orother tissues containing actively proliferating cells.

As a method well known by a person skilled in the art for preparing aprotein functionally equivalent to a given protein, a method forintroducing mutation into the protein is known. For example, one skilledin the art can prepare proteins functionally equivalent to the human“fls353” or “fls485” protein by introducing an appropriate mutation inthe amino acid sequence of the human “fls353” or “fls485” protein bysite-directed mutagenesis (Hashimoto-Gotoh et al., Gene, 152:271-275,1995; Zoller et al., Methods Enzymol., 100:468-500, 1983; Kramer et al.,Nucleic Acids Res., 12:9441-9456, 1984; Kramer et al., Methods.Enzymol., 154:350-367, 1987; Kunkel, Proc. Natl. Acad. Sci. USA,82:488-492, 1985; Kunkel, Methods Enzymol., 85:2763-2766, 1988).Mutation of amino acids could occur in nature, too. The protein of thepresent invention includes those comprising amino acid sequences of thehuman “fls353 or “fls485” protein in which one or more amino acids aremutated and functionally equivalent to the human “fls353” or “fls485”protein. It is considered that the number of amino acids to be mutatedin such a mutant, is generally 10 amino acids or less, preferably 5amino acids or less, and more preferably 3 amino acids or less. Proteinshaving amino acid sequences modified by deleting, adding and/orreplacing one or more amino acid residues of a certain amino acidsequence, have been known to retain the original biological activity(Mark et al., Proc. Natl. Acad. Sci. USA, 81:5662-5666, 1984; Zoller etal., Nucleic Acids Res., 10:6487-6500, 1982; Wang et al., Science,224:1431-1433; Dalbadie-McFarland et al., Proc. Natl. Acad. Sci. USA,79:6409-6413, 1982).

As for the amino acid residue to be mutated, it is preferable to bemutated into a different amino acid in which the properties of the aminoacid side-chain are conserved. Examples of properties of amino acid sidechains are, hydrophobic amino acids (A, I, L, M, F, P, W, Y, V),hydrophilic amino acids (R, D, N, C, E, Q, Q, H, K, S, T), and aminoacids comprising the following side chains: an aliphatic side-chain (G,A, V, L, I, P); a hydroxyl group containing side-chain (S, T, Y); asulfur atom containing side-chain (C, M); a carboxylic acid and amidecontaining side-chain (D, N, E, Q); a base containing side-chain (R, K,H); and an aromatic containing side-chain (H, F, Y, W). (The parentheticletters indicate the one-letter codes of amino acids.)

As a protein to which one or more amino acids residues are added to theamino acid sequence of human “fls353” or “fls485” protein (SEQ ID NOs:2,4 or 6), for example, a fusion protein comprising human “fls353” or“fls485” protein can be used. Fusion proteins are fusions of the human“fls353” or “fls485” protein and other peptides or proteins, and areincluded in the present invention. Fusion proteins can be made bytechniques well known to a person skilled in the art, such as by linkingthe DNA encoding the human “fls353” or “fls485” protein of the inventionwith DNA encoding other peptides or proteins, so as the frames match,inserting this into an expression vector and expressing it in a host.There is no restriction as to the peptides or proteins fused to theprotein of the present invention.

Known peptides, for example, FLAG (Hopp et al., Biotechnology,6:1204-1210, 1988), 6×His consisting of six His (histidine) residues,10×His, Influenza agglutinin (HA), human c-myc fragment, VSV-GPfragment, p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigenfragment, lck tag, α-tubulin fragment, B-tag, Protein C fragment, andsuch, can be used as peptides that are fused to the protein of thepresent invention. Examples of proteins that are fused to protein of thepresent invention are, GST (glutathione-S-transferase), Influenzaagglutinin (HA), immunoglobulin constant region, β-galactosidase, MBP(maltose-binding protein), and such.

Fusion proteins can be prepared by fusing commercially available DNAencoding these peptides or proteins with the DNA encoding the protein ofthe present invention and expressing the fused DNA prepared.

An alternative method known in the art to isolate functionallyequivalent proteins is, for example, the method using a hybridizationtechnique (Sambrook et al., Molecular Cloning 2nd ed., 9.47-9.58, ColdSpring Harbor Lab. Press, 1989). One skilled in the art can readilyisolate DNA having high homology with a whole or part of the DNAsequence (SEQ ID NOs:1, 3 or 5) encoding the human “fls353” or “fls485”protein, and isolate functionally equivalent proteins to the human“fls353” or “fls485” protein from the isolated DNA. The proteins of thepresent invention include those encoded by DNA that hybridizes with awhole or part of the DNA sequence encoding human “fls353” or “fls485”protein and functionally equivalent to the human “fls353” or “fls485”protein. These proteins include a homologue of mammals corresponding tothe protein derived from human (for example, a protein encoded by amonkey, rat, mouse, rabbit and bovine gene). When a cDNA highlyhomologous with the DNA encoding human “fls353” or “fls485” protein isisolated from animals, especially fetal tissues, fetal liver or kidneyparticularly preferable.

As used herein, an “isolated nucleic acid” is a nucleic acid, thestructure of which is not identical to that of any naturally occurringnucleic acid or to that of any fragment of a naturally occurring genomicnucleic acid spanning more than three genes. The term therefore covers,for example, (a) a DNA which has the sequence of part of a naturallyoccurring genomic DNA molecule but is not flanked by both of the codingsequences that flank that part of the molecule in the genome of theorganism in which it naturally occurs; (b) a nucleic acid incorporatedinto a vector or into the genomic DNA of a prokaryote or eukaryote in amanner such that the resulting molecule is not identical to anynaturally occurring vector or genomic DNA; (c) a separate molecule suchas a cDNA, a genomic fragment, a fragment produced by polymerase chainreaction (PCR), or a restriction fragment; and (d) a recombinantnucleotide sequence that is part of a hybrid gene, i.e., a gene encodinga fusion protein. Specifically excluded from this definition are nucleicacids present in random, uncharacterized mixtures of different DNAmolecules, transfected cells, or cell clones, e.g., as these occur in aDNA library such as a cDNA or genomic DNA library.

Accordingly, in one aspect, the invention provides an isolated orpurified nucleic acid molecule that encodes a polypeptide describedherein or a fragment thereof. Preferably, the isolated nucleic acidmolecule includes a nucleotide sequence that is at least 60% identicalto the nucleotide sequence shown in SEQ ID NOs:1, 3 or 5. Morepreferably, the isolated nucleic acid molecule is at least 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore, identical to the nucleotide sequence shown in SEQ ID NOs:1, 3 or5. In the case of an isolated nucleic acid molecule which is longer thanor equivalent in length to the reference sequence, e.g., SEQ ID NOs:1, 3or 5, the comparison is made with the full length of the referencesequence. Where the isolated nucleic acid molecule is shorter that thereference sequence, e.g., shorter than SEQ ID NOs:1, 3 or 5, thecomparison is made to a segment of the reference sequence of the samelength (excluding any loop required by the homology calculation).

As a condition of hybridization for isolating a DNA encoding a proteinfunctionally equivalent to human “fls353” or “fls485” protein, a personskilled in the art can conveniently select. One example of thehybridization condition for isolating functionally equivalent proteinsis as follows. That is, after prehybridization at 55° C. for 30 minutesor more, hybridization is performed by adding labeled probes andincubating at 37 to 55° C. for an hour or more using “ExpressHybHybridization Solution” (CLONTECH). After that, the resulting hybridizedproduct is washed three times for 20 minutes each at room temperature in2×SSC and 0.1% SDS then once at 37° C. in 1×SSC and 0.1% SDS.

More preferably (more stringently), after prehybridization at 60° C. for30 minutes or more, hybridization is performed by adding labeled probesand incubating at 60° C. for an hour or longer using “ExpressHybHybridization Solution” (CLONTECH). Thereafter, the hybridized productis washed three times for 20 minutes each at room temperature in 2×SSCand 0.1% SDS then twice at 50° C. in 1×SSC and 0.1% SDS.

Still more preferably (still more stringently), after prehybridizationat 68° C. for 30 minutes or more, hybridization is performed by addinglabeled probes and incubating at 68° C. for an hour or more using“ExpressHyb Hybridization Solution” (CLONTECH). Thereafter, thehybridized product is washed three times for 20 minutes each at roomtemperature in 2×SSC and 0.1% SDS then twice at 50° C. in 0.1×SSC and0.1% SDS.

However, several factors such as temperature or salt concentration caninfluence the stringency of hybridization and one skilled in the art cansuitably select the factors to accomplish a similar stringency. In placeof hybridization, the gene amplification method using a primersynthesized based on the sequence information of the DNA (SEQ ID NOs:1,3 or 5) encoding the human “fls353” or “fls485” protein, for example,the polymerase chain reaction (PCR) method can be utilized to isolate aDNA encoding the human “fls353” or “fls485” protein.

Proteins that have functionally equivalent as human “fls353” or “fls485”protein encoded by the DNA isolated through the above hybridizationtechnique or gene amplification techniques, usually have a high homologyto the amino acid sequence of the human “fls353” or “fls485” protein.“High homology” refers to, usually a homology of 60% or higher,preferably 70% or higher, more preferably 80% or higher, even morepreferably 90% or higher. The homology of a protein can be determined byfollowing the algorithm in “Wilbur et al., Proc. Natl. Acad. Sci. USA,80:726-730, 1983”.

As used herein, “% identity” of two amino acid sequences, or of twonucleic acid sequences, is determined using the algorithm of Karlin andAltschul (Proc. Natl. Acad. Sci. USA, 87:2264-2268, 1990), modified asin Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5877, 1993).Such an algorithm is incorporated into the NBLAST and XBLAST programs ofAltschul et al. (J. Mol. Biol., 215:403-410, 1990). BLAST nucleotidesearches are performed with the NBLAST program, score=100,wordlength=12. BLAST protein searches are performed with the XBLASTprogram, score=50, wordlength=3. To obtain gapped alignment forcomparison purposes GappedBLAST is utilized as described in Altschul etal. (Nucleic Acids Res., 25:3389-3402, 1997). When utilizing BLAST andGappedBLAST programs the default parameters of the respective programs(e.g., XBLAST and NBLAST) are used to obtain nucleotide sequenceshomologous to a nucleic acid molecule of the invention.

A protein of the present invention may have variations in amino acidsequence, molecular weight, isoelectric point, the presence or absenceof sugar chains, or form, depending on the cell or host used to produceit or the purification method utilized. Nevertheless, as long as it hassimilar function as human “fls353” or “fls485” protein (SEQ ID NOs:2, 4or 6) of the present invention, it is within the scope of the presentinvention.

The proteins of the present invention can be prepared as a recombinantprotein or a natural protein by the method well known by the personskilled in the art. A recombinant DNA can be prepared by inserting a DNA(for example, the DNA comprising the nucleotide sequence of SEQ IDNOs:1, 3 or 5) which codes the protein of the present invention into anappropriate vector, collecting a recombinant obtained by introducing thevector into appropriate host cells, obtaining the extract, and purifyingby subjecting the extract to chromatography such as ion exchange,reverse, gel filtration, or affinity chromatography in which an antibodyagainst the protein of the present invention is fixed on column or bycombining more than one of these columns.

Also when the protein of the present invention is expressed within hostcells (for example, animal cells and E. coli) as a fusion protein withglutathione-S-transferase protein or as a recombinant proteinsupplemented with multiple histidines, the expressed recombinant proteincan be purified using a glutathione column or nickel column.

After purifying the fusion protein, it is also possible to excluderegions other than the objective protein by cutting with thrombin orfactor-Xa as required. A natural protein can be isolated by the methodknown by a person skilled in the art, for example, by effecting theaffinity column in which the antibody binding to the human “fls353” or“fls485” protein described below is bound against the extract of tissuesor cells expressing the protein of the present invention is expressed.An antibody can be a polyclonal or a monoclonal antibody.

The present invention also contains a partial peptide of the protein ofthe present invention. A partial peptide comprising the amino acidsequence specific to the protein of the present invention comprises atleast 7 amino acids, preferably 8 amino acids or more, and morepreferably 9 amino acids or more. The partial peptide can be used, forexample, for preparing an antibody against the protein of the presentinvention, screening a compound biding to the protein of the presentinvention, and for screening accelerators or inhibitors of the proteinof the present invention.

A partial peptide of the invention can be produced by geneticengineering, known methods of peptide synthesis, or by digesting theprotein of the invention with an appropriate peptidase. For peptidesynthesis, for example, solid phase synthesis or liquid phase synthesismay be used.

Furthermore, the present invention relates to a DNA encoding the proteinof the present invention as described. The DNA of the present inventioncan be used for the production of the protein of the present inventionin vivo or in vitro as described above as well as for, for example,application to the gene therapy for diseases attributed to geneticabnormality in the gene encoding the protein of the present invention.Any form of the DNA of the present invention can be used as long as itencodes the protein of the present invention. Specifically, cDNAsynthesized from the mRNA, genomic DNA, or chemically synthesized DNAcan be used. The present invention includes a DNA comprising a givennucleotide sequence based on degeneracy of genetic codons, as long as itencodes the protein of the present invention.

The DNA of the present invention can be prepared by the method known bya person skilled in the art. For example, the DNA of the presentinvention can be prepared by preparing a cDNA library from cells whichexpress the protein of the present invention, and conductinghybridization by using a partial sequence of the DNA of the presentinvention (e.g., SEQ ID NOs:1, 3, or 5) as a probe. A cDNA library canbe prepared, for example, by the method described in Sambrook et al.,Molecular Cloning, Cold Spring Harbor Laboratory Press (1989), or byusing a cDNA library in the market. A cDNA library can be also preparedby preparing RNA from cells expressing the protein of the presentinvention, synthesizing an oligo DNA base on the sequence of the DNA ofthe present invention (for example, SEQ ID NOs:1, 3 or 5), conductingPCR by using these as primers, and amplifying cDNA encoding the proteinof the present invention.

In addition, by sequencing the nucleotides of the obtained cDNA, atranslation region encoded by this can be determined, and an amino acidsequence of the protein of the present invention can be obtained.Moreover, by screening the genomic DNA library using the obtained cDNAas a probe, genomic DNA can be isolated.

More specifically, mRNAs may first be prepared from a cell, tissue, ororgan (for example, fetal liver or kidney, carcinoma clone and so on) inwhich the protein of the invention is expressed. Known methods can beused to isolate mRNAs, for instance, total RNA is prepared by guanidineultracentrifugation (Chirgwin et al., Biochemistry, 18:5294-5299, 1979)or AGPC method (Chomczynski et al., Anal. Biochem., 162:156-159, 1987),and mRNA is purified from total RNA using mRNA Purification Kit(Pharmacia) and such. Alternatively, mRNA may be directly purified byQuickPrep mRNA Purification Kit (Pharmacia).

The obtained mRNA is used to synthesize cDNA using reversetranscriptase. cDNA may be synthesized by using a kit such as the AMVReverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Kogyo).Alternatively, cDNA may be synthesized and amplified following the5′-RACE method (Frohman et al., Proc. Natl. Acad. Sci. USA, 85:8998-9002, 1988); Belyaysky et al., Nucleic Acids Res., 17:2919-2932,1989) which uses a primer and such, described herein, the 5′-AmpliFINDER RACE Kit (CLONTECH), and polymerase chain reaction (PCR).

A desired DNA fragment is prepared from the PCR products and ligatedwith a vector DNA. The recombinant vectors are used to transform E. coliand such, and a desired recombinant vector is prepared from a selectedcolony. The nucleotide sequence of the desired DNA is able to verify byconventional methods, such as dideoxynucleotide chain termination.

A DNA of the invention may be designed to have a sequence that isexpressed more efficiently by taking into account the frequency of codonusage in the host to be used for expression (Grantham et al., NucleicAcids Res., 9:43-74, 1981). The DNA of the present invention may bealtered by a commercially available kit or a conventional method. Forinstance, the DNA may be altered by digestion with restriction enzymes,insertion of a synthetic oligonucleotide or an appropriate DNA fragment,addition of a linker, or insertion of the initiation codon (ATG) and/orthe stop codon (TAA, TGA, or TAG).

Specifically, DNAs of this invention include DNAs comprising the basesA472 through C2712 in the nucleotide sequence set forth in SEQ ID NO:1,the bases A247 through G1305 in the nucleotide sequence set forth in SEQID NO:3, and the bases A254 through G1159 in the nucleotide sequence setforth in SEQ ID NO:5.

Furthermore, the present invention provides DNA that is capable ofhybridizing with DNA having a nucleotide sequence of SEQ ID NOs:1, 3 or5 under stringent conditions, and encoding a protein functionallyequivalent to the protein of the invention described above.

Stringent conditions may be appropriately chosen by one skilled in theart, and, for example, low stringent conditions can be used. Morepreferably, high stringent conditions can be used. These conditions arethe same as the above. The above hybridizing DNA is preferably a cDNA orchromosomal DNA.

The present invention also relates to a vector into which the DNA of thepresent invention is inserted. The vector of the present invention isuseful to keep DNA of the present invention in host cell, or to expressthe protein of the present invention.

When E. coli is a host cell and the vector is amplified and produced ina large amount in E. coli (e.g., JM109, DH5α, HB101, or XL1Blue), thevector should have “ori” to be amplified in E. coli and a marker genefor selecting transformed E. coli (e.g., a drug-resistance gene selectedby a drug (e.g., ampicillin, tetracycline, kanamycin, orchloramphenicol)). For example, M13-Series vectors, pUC-series vectors,pBR322, pBluescript, pCR-Script, and so on can be used. For example,pGEM-T, pDIRECT, and pT7 can also be used for subcloning and extractingcDNA as well as the vectors described above. When a vector is used toproduce the protein of the present invention, an expression vector isespecially useful. For example, an expression vector to be expressed inE. coli should have the above characteristics to be amplified in E.coli. When E. coli, such as JM109, DH5α, HB101, or XL1 Blue, are used asa host cell, the vector should have a promoter as well as the abovecharacters such as the vector is copied in the host, for example, lacZpromotor (Ward et al., Nature, 341:544-546, 1989; FASEB J., 6:2422-2427,1992), araB promoter (Better et al., Science, 240:1041-1043, 1988), orT7 promoter and such, that can efficiently express the desired gene inE. coli. As such a vector, for example, pGFX-5X-1 (Pharmacia),“QIAexpress system” (Qiagen), pEGFP or pET (in this case, a host ispreferably BL21 which expresses T7 RNA polymerase) can be used besidesthe above vectors.

A vector also may contain a signal sequence for polypeptide secretion.As a signal sequence for protein secretion, pelB signal sequence (Lei etal., J. Bacteriol., 169:4379, 1987) can be used in the case of producingperiplasm in E. coli. For introducing a vector into host cells, forexample, calcium chloride method, and electroporation method can beused.

Besides E. coli, for example, expression vectors derived from mammals(for example, pcDNA3 (Invitrogen) and pEGF-BOS (Nucleic Acids Res.,18:5322, 1990), pEF, pCDM8), expression vectors derived from insectcells (for example, “Bac-to-Bac baculovairus expression system” (GIBCOBRL), pBacPAK8), expression vectors derived from plants (for example,pMH1, pMH2), expression vectors derived from animal viruses (forexample, pHSV, pMV, pAdexLcw), expression vectors derived fromretroviruses (for example, pZIPneo), expression vector derived fromyeast (for example, “Pichia Expression Kit” (Invitrogen), pNV11,SP-Q01), expression vectors derived from Bacillus subtilis (for example,pPL608, pKTH50) can be used for producing the protein of the presentinvention.

In order to express the vector in animal cells, such as CHO, COS, orNIH3T3 cells, the vector should have a promoter necessary for expressionin such cells, for example, SV40 promoter (Mulligan et al., Nature,277:108, 1979), MMLV-LTR promoter, EF1α promoter (Mizushima et al.,Nucleic Acids Res., 18:5322, 1990), or CMV promoter, and such, andpreferably a marker gene for selecting transformants (for example, adrug resistance gene selected by a drug (e.g., neomycin, G418)).Examples of the vectors with these characteristics include pMAM, pDR2,pBK-RSV, pBK-CMV, pOPRSV, and pOp13 and so on.

In addition, for the purpose of expressing a gene stably and amplifyingthe copy number of the gene in cells, for example, the method forintroducing a vector comprising the complementary DHFR gene (forexample, pCHOI), into CHO cells in which nuclei acid synthesizingpathway is deleted and amplifying by methotrexate (MTX) can be used andin the case of transient expression of a gene, the method fortransforming with a vector (e.g., pcD) comprising replication origin ofSV40 using COS cells comprising the SV40 T antigen expressing gene onchromosomes can be used.

On the other hand, the DNA of the present invention can be expressed invivo in animals, for example, by inserting the DNA of the presentinvention into an appropriate vector and introducing in vivo by such asretrovirus method, liposome method, cationic liposome method, adenovirusmethod. By using these, gene therapy against diseases attributed tomutation of “fls353” or “fls485” gene of the present invention can beeffected. As a vector to be used, for example, adenovirus vector (forexample, pAdexlcw), and retrovirus vector (for example, pZIPneo) can beused, but not restricted thereto. Common gene manipulation, for example,insertion of the DNA of the present invention to a vector, can beperformed according to the standard method (Sambrook, J. et al.,Molecular Cloning 2nd ed., 5. 61-5. 63, Cold Spring Harbor Lab. Press,1989). Administration into a living body can be either ex vivo method,or in vivo method.

The present invention relates to a host cell into which the vector ofthe present invention has been introduced. The host cell into which thevector of the invention is introduced is not particularly limited. E.coli or various animal cells can be used. The host cells of the presentinvention can be used for, for example, production system for producingor expressing the protein of the present invention. The presentinvention provides methods of producing a protein of the invention bothin vitro or in vivo. For in vitro production, eukaryotic cells orprokaryotic cells can be used as host cells.

Useful eukaryotic cells as host may be animal, plant, or fungi cells. Asanimal cells, mammalian cells such as CHO (J. Exp. Med., 108:945, 1995),COS, 3T3, myeloma, baby hamster kidney (BHK), HeLa, Vero cells, oramphibian cells such as Xenopus oocytes (Valle, et al., Nature,291:340-358, 1981), or insect cells such as sf9, sf21, or Tn5 cells canbe used. CHO cells lacking DHFR gene (dhfr-CHO) (Proc. Natl. Acad. Sci.USA, 77:4216-4220, 1980) or CHO K-1 (Proc. Natl. Acad. Sci. USA,60:1275, 1968) may also be used. In animal cells, CHO cells areparticularly preferable for the mass expression. A vector can beintroduced into host cells by, for example, calcium phosphate method,DEAE dextran method, cationic liposome DOTAP (Boehringer Mannheim),electroporation method, lipofection method. As plant cells, plant cellsoriginating from Nicotiana tabacum are known as protein-productionsystem, and may be used as callus cultures. As fungi cells, yeast cellssuch as Saccharomyces, including Saccharomyces cerevisiae, orfilamentous fungi such as Aspergillus, including Aspergillus niger, areknown and may be used herein.

Useful prokaryotic cells include bacterial cells, such as E. coli, forexample, JM109, DH5α, HB101 are known. Others, Bacillus subtilis isknown.

These cells are transformed by a desired DNA, and the resultingtransformants are cultured in vitro to obtain the protein. Transformantscan be cultured using known methods. Culture medium for animal cell, forexample, DMEM, MEM, RPMI1640, or IMDM may be used with or without serumsupplement such as fetal calf serum (FCS). The pH of the culture mediumis preferably between about pH 6 to 8. Such cells are typically culturedat about 30 to 40° C. for about 15 to 200 hr, and the culture medium maybe replaced, aerated, or stirred if necessary.

Animal and plant hosts may be used for in vivo production. For example,a desired DNA can be introduced into an animal or plant host. Encodedproteins are produced in vivo, and then recovered. These animal andplant hosts are included in host cells of the present invention.

Animals to be used for the production system described above include,but are not limited to, mammals and insects. Mammals such as goat,porcine, sheep, mouse, and bovine may be used (Vicki Glaser, SPECTRUMBiotechnology Applications (1993)). Alternatively, the mammals may betransgenic animals.

For instance, a desired DNA may be prepared as a fusion gene with a geneencoding a protein specifically produced into milk, such as goat βcasein. DNA fragments comprising the fusion gene having the desired DNAare injected into goat embryos, which are then introduced back to femalegoats. Proteins are recovered from milk produced by the transgenic goats(i.e., those born from the goats that had received the modified embryos)or from their offspring. To increase the amount of milk containing theproteins produced by transgenic goats, appropriate hormones may beadministered to them (Ebert et al., Bio/Technology, 12:699-702, 1994).

Alternately, insects, such as the silkworm, may be used. A desired DNAinserted into baculovirus can be used to infect silkworms, and thedesired protein is recovered from their body fluid (Susumu et al.,Nature, 315:592-594, 1985).

As plants, for example, tobacco can be used. In use of tobacco, adesired DNA may be inserted into a plant expression vector, such aspMON530, which is introduced into a bacteria, such as Agrobacteriumtumefaciens. Then the bacteria is used to infect tobacco, such asNicotiana tabacum, and a desired polypeptide is recovered from theirleaves (Julian et al., Eur. J. Immunol., 24:131-138, 1994).

A protein of the present invention obtained as above may be isolatedfrom inside or outside (e.g., medium) of cells or hosts, and purified assubstantially pure homogeneous protein. The method for protein isolationand purification is not limited to any specific method; in fact, anystandard method may be used. For instance, column chromatography,filter, ultrafiltration, salt precipitation, solvent precipitation,solvent extraction, distillation, immunoprecipitation,SDS-polyacrylamide gel electrophoresis, isoelectric pointelectrophoresis, dialysis, and recrystallization may be appropriatelyselected and combined to isolate and purify the protein.

For chromatography, for example, affinity chromatography, ion-exchangechromatography, hydrophobic chromatography, gel filtration, reversephase chromatography, adsorption chromatography, and such may be used(Strategies for Protein Purification and Characterization: A LaboratoryCourse Manual. Ed, Daniel R. Marshak et al., Cold Spring HarborLaboratory Press (1996)). These chromatographies may be performed byliquid chromatography such as HPLC and FPLC. Thus, the present inventionprovides for highly purified proteins, produced by the above methods.

A protein of the present invention may be optionally modified orpartially deleted by treating it with an appropriate proteinmodification enzyme before or after purification. Useful proteinmodification enzymes include, but are not limited to, trypsin,chymotrypsin, lysylendopeptidase, protein kinase, and glucosidase.

The present invention relates to an antibody that binds to the proteinof the invention. The antibody of the invention can be used in any form,such as monoclonal or polyclonal antibodies, and includes antiserumobtained by immunizing a rabbit with the protein of the invention, allclasses of polyclonal and monoclonal antibodies, human antibodies, andhumanized antibodies produced by genetic recombination.

A protein of the invention used as an antigen to obtain an antibody maybe derived from any animal species, but preferably from a mammal such asa human, mouse, or rat, or more preferably from a human. A human-derivedprotein may be obtained from the nucleotide or amino acid sequencesdisclosed herein.

In the present invention, a protein to be used as an immunizationantigen may be a complete protein or a partial peptide of the protein. Apartial peptide may be, for example, an amino (N)-terminal or carboxy(C)-terminal fragment of the protein. Herein, “an antibody” is definedas an antibody that specifically reacts with the full length or afragment of the protein.

A gene encoding a protein of the invention or its fragment may beinserted into a known expression vector, which is used to transform ahost cell as described herein. The desired protein or its fragment maybe recovered from the outside or inside of host cells by any standardmethod, and may be used as an antigen. Alternatively, cells expressingthe protein or their lysates, or a chemically synthesized protein may beused as an antigen.

Any mammalian animal may be immunized with the antigen, but preferablythe compatibility with parental cells used for cell fusion is taken intoaccount. In general, animals of Rodentia, Lagomorphs, or Primates areused.

Animals of Rodentia include, for example, mouse, rat, and hamster.Animals of Lagomorphs include, for example, rabbit. Animals of Primatesinclude, for example, a monkey of catarrhine (old world monkey) such asMacaca fascicularis, rhesus monkey, sacred baboon, or chimpanzee.

Methods for immunizing animals against antigens are known in the art.Intraperitoneal injection or subcutaneous injection of antigens is usedas a standard method for immunization of mammals. More specifically,antigens may be diluted and suspended in an appropriate amount withphosphate buffered saline (PBS), physiological saline, etc. If desired,the antigen suspension may be mixed with an appropriate amount of astandard adjuvant, such as Freund's complete adjuvant, made intoemulsion, and then administered to mammalian animals. Preferably, it isfollowed by several administrations of antigen mixed with anappropriately amount of Freund's incomplete adjuvant every 4 to 21 days.An appropriate carrier may also be used for immunization. Afterimmunization as above, serum is examined for increase of the amount ofdesired antibodies by a standard method.

Polyclonal antibodies against the proteins of the present invention maybe prepared by collecting blood from the immunized mammal examined forthe increase of desired antibodies in the serum, and by separating serumfrom the blood by any conventional method. Polyclonal antibodies may beused as serum containing the polyclonal antibodies, or if necessary, afraction containing the polyclonal antibodies may be isolated from theserum. Immunoglobulin G or M can be prepared by obtaining a fractionwhich recognizes only the protein of the present invention using anaffinity column coupled with the protein of the present invention andfurther purifying this fraction by using protein A or protein G column.

To prepare monoclonal antibodies, immune cells are collected from themammal immunized against the antigen and checked for the increased levelof desired antibodies in the serum as described above, and are subjectedto cell fusion. The immune cells used for cell fusion are preferablyobtained from spleen. As the other parental cells to be fused with theabove immunocyte, for example, preferably myeloma cells of mammalians,and more preferably myeloma cells which acquired the property forselecting fused cells by drugs can be used.

The above immunocyte and myeloma cells can be fused by the known method,for example, the method by Milstein et al. (Methods Enzymol., 73:3-46,1981).

Resulting hybridomas obtained by the cell fusion may be selected bycultivating them in a standard selection medium, such as HAT medium(medium containing hypoxanthine, aminopterin, and thymidine). The cellculture is typically continued in the HAT medium for several days toseveral weeks, the sufficient time to allow all the other cells, exceptdesired hybridoma (non-fused cells), to die. Then, by the standardlimiting dilution method, a hybridoma cell producing the desiredantibody is screened and cloned.

Besides the above method, in which a non human animal is immunizedagainst an antigen for preparing hybridoma, human lymphocytes such asthat infected by EB virus may be immunized with a protein, proteinexpressing cells, or their lysates in vitro. Then, the immunizedlymphocytes are fused with human-derived myeloma cells capable ofindefinitely dividing, such as U266, to yield a hybridoma producing adesired human antibody having binding ability to the protein can beobtained (Unexamined Published Japanese Patent Application (JP-A) No.Sho 63-17688).

Next, the monoclonal antibody obtained by transplanting the obtainedhybridomas into the abdominal cavity of a mouse and by extractingascites can be purified by, for example, ammonium sulfate precipitation,protein A or protein G column, DEAE ion exchange chromatography, or anaffinity column to which the protein of the present invention iscoupled. The antibody of the present invention can be used not only forpurification and detection of the protein of the present invention, butalso as a candidate for agonists and antagonists of the protein of thepresent invention. In addition, this antibody can be applied to theantibody treatment for diseases involved by the protein of the presentinvention. When the obtained antibody is used for the administration tothe human body (antibody treatment), a human antibody or a humanizedantibody is preferable for reducing immunogenicity.

For example, transgenic animals having a repertory of human antibodygenes may be immunized against a protein, protein expressing cells, ortheir lysates as an antigen. Antibody producing cells are collected fromthe animals, and fused with myeloma cells to obtain hybridoma, fromwhich human antibodies against the protein can be prepared (seeWO92-03918, WO93-2227, WO94-02602, WO94-25585, WO96-33735, andWO96-34096)

Alternatively, an immune cell, such as an immunized lymphocyte,producing antibodies may be immortalized by an oncogene and used forpreparing monoclonal antibodies.

Monoclonal antibodies thus obtained can be also recombinantly preparedusing genetic engineering techniques (see, for example, Borrebaeck C. A.K. and Larrick, J. W., THERAPEUTIC MONOCLONAL ANTIBODIES, published inthe United Kingdom by MACMILLAN PUBLISHERS LTD (1990)). A DNA encodingan antibody may be cloned from an immune cell such as a hybridoma or animmunized lymphocyte producing the antibody, inserted into anappropriate vector, and introduced into host cells to prepare arecombinant antibody. The present invention also provides recombinantantibodies prepared as described above.

Furthermore, an antibody of the present invention may be a fragment ofan antibody or modified antibody, so long as it binds to one or more ofthe proteins of the invention. For instance, the antibody fragment maybe Fab, F (ab′)₂, Fv, or single chain Fv (scFv), in which Fv fragmentsfrom H and L chains are ligated by an appropriate linker (Huston et al.,Proc. Natl. Acad. Sci. USA, 85:5879-5883, 1988). More specifically, anantibody fragment may be generated by treating an antibody with anenzyme such as papain or pepsin. Alternatively, a gene encoding theantibody fragment may be constructed, inserted into an expressionvector, and expressed in an appropriate host cell (see, for example, Coet al., J. Immunol., 152:2968-2976, 1994; Better et al., MethodsEnzymol., 178:476-496, 1989; Pluckthun et al., Methods Enzymol.,178:497-515, 1989; Lamoyi, E., Methods Enzymol., 121:652-663, 1986;Rousseaux et al., Methods Enzymol., 121:663-669, 1986; Bird et al.,Trends Biotechnol., 9:132-137, 1991).

An antibody may be modified by conjugation with a variety of molecules,such as polyethylene glycol (PEG). The present invention provides suchmodified antibodies. The modified antibody can be obtained by chemicallymodifying an antibody. These modification methods are conventional inthis field.

Alternatively, an antibody of the present invention may be obtained as achimeric antibody, between a variable region derived from nonhumanantibody and the constant region derived from human antibody, or as ahumanized antibody, comprising the complementarity determining region(CDR) derived from nonhuman antibody, the frame work region (FR) derivedfrom human antibody, and the constant region.

Obtained antibodies may be purified into homogeneity. The antibody usedin the present invention can be separated and purified by used forseparating and purifying usual proteins. For example, the separation andpurification of the protein can be performed by the appropriatelyselected and combined use of column chromatography such as affinitychromatography, filter, ultrafiltration, salting-out, dialysis, SDSpolyacrylamide gel electrophoresis, isoelectric focusing, and others(Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold SpringHarbor Laboratory, 1988), but the methods are not limited thereto.

Examples of columns used for affinity chromatography include protein Acolumn and protein G column. Examples of columns using protein A columninclude Hyper D, POROS, Sepharose F. F, (Pharmacia), etc.

Besides affinity chromatography, the chromatography includes, forexample, ion-exchange chromatography, hydrophobic chromatography, gelfiltration, reverse-phase chromatography, adsorption chromatography, andthe like (Strategies for Protein Purification and Characterization: ALaboratory Course Manual. Ed Daniel R. Marshak et al, Cold Spring HarborLaboratory Press. 1996). The chromatographic procedures can be carriedout by liquid-phase chromatography such as HPLC, FPLC, or the like.

For example, measurement of absorbance, enzyme-linked immunosorbentassay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), and/orimmunofluorescence may be used to measure the antigen binding activityof the antibody of the invention. In ELISA, the antibody of the presentinvention is immobilized on a plate, protein of the invention is appliedto the plate, and then a sample containing a desired antibody, such asculture supernatant of antibody producing cells or purified antibodies,is applied. Then, a secondary antibody, that recognizes the primaryantibody and is labeled with an enzyme such as alkaline phosphatase, isapplied, and the plate is incubated. Next, after washing, an enzymesubstrate, such as p-nitrophenyl phosphate, is added to the plate, andthe absorbance is measured to evaluate the antigen binding activity ofthe sample. A fragment of the protein, such as a C-terminal orN-terminal fragment, may be used as a protein. BIAcore (Pharmacia) maybe used to evaluate the activity of the antibody according to thepresent invention.

The above methods allow for the detection or measurement of the proteinof the invention, by exposing the antibody of the invention to a sampleassumed to contain the protein of the invention, and detecting ormeasuring the immune complex formed by the antibody and the protein.

Because the method of detection or measurement of proteins according tothe invention can specifically detect or measure proteins, the methodmay be useful in a variety of experiments in which the protein is used.

The present invention relates a nucleotide which hybridizes with the DNAencoding human “fls353” or “fls485” protein (SEQ ID NOs:1, 3 or 5) orthe complementary strand, and comprises at least 15 nucleotides. Thenucleotide of the present invention is preferably the nucleotide whichspecifically hybridizes with the DNA encoding the protein of the presentinvention. The term “specifically hybridize” as used herein, means thatcross-hybridization does not occur significantly with DNA encoding otherproteins, in the above-mentioned hybridizing conditions, preferablyunder stringent hybridizing conditions. Such nucleotide includes,probes, primers, nucleotides and nucleotide derivatives (for example,antisense oligonucleotides and ribozymes and so on), which specificallyhybridize with DNA encoding the protein of the invention or itscomplementary strand. Moreover, such nucleotide can utilize aspreparation of DNA chip.

The present invention includes an antisense oligonucleotide thathybridizes with any site within the nucleotide sequence any one of SEQID NOs:1, 3 and 5. This antisense oligonucleotide is preferably thatagainst the at least 15 continuous nucleotides in the nucleotidesequence any one of SEQ ID NOs: 1, 3 or 5. The above-mentioned antisenseoligonucleotide, which contains an initiation codon in theabove-mentioned at least 15 continuous nucleotides, is even morepreferred.

Derivatives or modified products of antisense oligonucleotides can beused as antisense oligonucleotides. Examples of such modified productsare, lower alkyl phosphonate modifications such asmethyl-phosphonate-type or ethyl-phosphonate-type, phosphothioatemodifications and phosphoamidate modifications.

The term “antisense oligonucleotides” as used herein means, not onlythose in which the entire nucleotides corresponding to thoseconstituting a specified region of a DNA or mRNA are complementary, butalso those having a mismatch of one or more nucleotides, as long as DNAor mRNA and an oligonucleotide can specifically hybridize with thenucleotide sequence of SEQ ID NOs:1, 3 or 5.

Such nucleotides have a homology of at least 70%, preferably at least80%, more preferably 90% or higher, even more preferably 95% or higherin the at least 15 continuous nucleotide sequence region. The algorithmstated herein can be used to determine homology. Such oligonucleotidesare useful as probes for the isolation or detection of DNA encoding theprotein of the invention as stated in a later example or as a primerused for amplifications.

The antisense oligonucleotide derivative of the present invention hasinhibitory effect on the function of the protein of the presentinvention as a result that the derivative inhibits the expression of theprotein of the invention by acting upon cells producing the protein ofthe invention and by binding to the DNA or mRNA encoding the protein toinhibit its transcription or translation or to promote the degradationof the mRNA.

The antisense oligonucleotide derivative of the present invention can bemade into an external preparation such as a liniment and a poultice bymixing with a suitable base material which is inactive against thederivatives.

Also, as necessary, the derivatives can be formulated into tablets,powders, granules, capsules, liposome capsules, injections, solutions,nose-drops, and freeze-drying agents and such by adding excipients,isotonic agents, solubilizing agents, stabilizers, preservativesubstance, pain-killers, and such. These can be prepared by followingusual methods.

The antisense oligonucleotide derivative is given to the patient bydirectly applying onto the ailing site or by injecting into a bloodvessel so that it will reach the site of ailment. An antisense-mountingmedium can also be used to increase durability andmembrane-permeability. Examples are, liposome, poly-L-lysine, lipid,cholesterol, lipofectin or derivatives of these.

The dosage of the antisense oligonucleotide derivative of the presentinvention can be adjusted suitably according to the patient's conditionand used in desired amounts. For example, a dose range of 0.1 to 100mg/kg, preferably 0.1 to 50 mg/kg can be administered.

The antisense oligonucleotide of the invention inhibits the expressionof the protein of the invention and thereby useful for suppressing thebiological activity of the protein of the invention. Also,expression-inhibitors comprising the antisense oligonucleotide of theinvention are useful in that they can inhibit the biological activity ofthe protein of the invention. It is thought it possible to use antisenseoligonucleotides of this invention for the purpose of antineoplastic.

Moreover the present invention relates to a method for screening acompound binding to the protein of the present invention by using theprotein of the present invention. This screening method contains thesteps of: (a) contacting a test sample with the protein of the presentinvention or a partial peptide thereof, (b) detecting a binding activityof the test sample to the protein of the present invention or thepartial peptide thereof, and (c) selecting a compound which binds to theprotein of the present invention or the partial peptide thereof.

The protein of the present invention to be used for screening may be arecombinant protein, a protein derived from the nature, or the partialpeptide thereof. Any test sample, for example, cell extracts, cellculture supernatant, products of fermenting microorganism, extracts frommarine organism, plant extracts, purified or crude proteins, peptides,non-peptide compounds, synthetic low molecular compounds and naturalcompounds, can be used. The protein of the present invention to becontacted with a test sample can be contacted, for example, as apurified protein, a soluble protein, a form bound to a carrier, or afusion protein with another protein.

By using the protein of the present invention, for example, as a methodfor screening a protein binding to the protein thereof, many methodswell known by a person skilled in the art can be used. Such a screeningcan be conducted by, for example, immunoprecipitation method,specifically, in the following manner. The gene encoding the protein ofthe present invention is expressed in such as animal cells by insertingthe gene to a expression vector for foreign gene, such as pSV2neo, pcDNAI, pCD8. As a promoter to be used for the expression, any promoter whichcan be used in general can be selected, for example, SV40 early promoter(Rigby in Williamson (ed.), Genetic engineering, vol. 3. Academic Press,London, p. 83-141 (1982)), EF-1α promoter (Kim et al., Gene, 91:217-223,1990), CAG promoter (Niwa et al., Gene, 108:193-200, 1991), RSV LTRpromoter (Cullen, Methods in Enzymology, 152:684-704, 1987), SRαpromoter (Takebe et al, Mol. Cell. Boil., 8:466, 1988), CMV immediateearly promoter (Seed et al., Proc. Natl. Acad. Sci. USA, 84:3365-3369,1987), SV40 late promoter (Gheysen et al., J. Mol. Appl. Genet.,1:385-394, 1982), Adenovirus late promoter (Kaufman et al., Mol. Cell.Biol., 9:946, 1989), HSV TK promoter and so on. To express a foreigngene by introducing the gene into animal cells, electroporation method(Chu et al., Nucl. Acid Res., 15:1311-1326, 1987), calcium phosphatemethod (Chen et al., Mol. Cell. Biol., 7:2745-2752, 1987), DEAE dextranmethod (Lopata et al., Nucl. Acids Res., 12:5707-5717, 1984; Sussman, etal., Mol. Cell. Biol., 4:1642-1643, 1985), Lipofectin method (Derijard,Cell, 7:1025-1037, 1994; Lamb et al., Nature Genetics, 5:22-30, 1993;Rabindran et al., Science, 259:230-234, 1993), and such can beexemplified, and any method can be used. The protein of the presentinvention can be expressed as a fusion protein comprising an arecognition site (epitope) of monoclonal antibody by introducing theepitope of the monoclonal antibody which property has been revealed to Nor C terminus of the protein of the present invention. Anepitope-antibody system in the market can be used (ExperimentalMedicine, 13:85-90, 1995). Through a multiple cloning site, a vectorwhich can express a fusion protein with, for example, β-galactosidase,maltose binding protein, glutathione S-transferase, green florescenceprotein (GFP), is available in the market.

Methods have been reported in which a fusion protein is prepared byintroducing only small epitopes comprising several to ten and severalamino acids so that properties of the protein of the present inventionmay not change by making the protein a fusion protein. Epitopes, forexample, polyhistidine (His-tag), influenza aggregate HA, human c-myc,FLAG, Vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10protein (T7-tag), human simple herpes virus glycoprotein (HSV-tag),epitope such as E-tag (an epitope on monoclonal phage), and monoclonalantibodies recognizing these can be, used as an epitope-antibody systemfor screening a protein binding to the protein of the present invention(Experimental Medicine, 13:85-90, 1995).

In the immunoprecipitation, an immune complex is formed by adding theseantibodies to cell eluate prepared by using an appropriate detergent.This immune complex comprises the protein of the present invention, aprotein comprising the binding ability with the protein, and anantibody. Immunoprecipitation can be conducted by an antibody againstthe protein of the present invention, besides using antibodies againstthe above epitopes. An antibody against the protein of the presentinvention can be prepared, for example by introducing a gene encodingthe protein of the present invention to an appropriate E. coliexpression vector, expressing the gene in E. coli, purifying theexpressed protein, and immunizing the protein to for example, rabbits,mice, rats, goats, and domestic fowls and such. The antibody can beprepared also by immunizing the above animals against a synthesizedpartial peptide of the protein of the present invention.

An immune complex can be precipitated, for example by Protein ASepharose or Protein G Sepharose when an antibody is mouse IgG antibody.When the protein of the present invention is prepared as a fusionprotein with an epitope, for example GST, an immune complex can beformed by using a substance specifically binding to these epitopes, suchas glutathione-Sepharose 4B in the same manner as in the use of anantibody of the protein of the present invention.

Popular Immunoprecipitation can be performed by following or accordingto, for example, the reference (Harlow et al., Antibodies, 511-552, ColdSpring Harbor Laboratory publications, New York (1988)).

SDS-PAGE is commonly used for analysis of immunoprecipitated proteinsand the binding protein can be analyzed depending on a molecular weightof the protein by using gel with an appropriate concentration. Ingeneral, because it is difficult to detect a protein binding to theprotein of the present invention by a common staining method, such asCoomassie staining or silver staining, the detection sensitivity for theprotein can be improved by culturing in the culture medium containingradioactive isomer, ³⁵S-methionine or ³⁵S-cystein, labeling proteins inthe cells, and detecting the proteins. The target protein can bepurified from SDS-polyacrylamide gel and its sequence can be determineddirectly after a molecular weight of the protein is determined.

As a method for isolating a protein binding to the protein of thepresent invention by using the protein, for example, West-Westernblotting analysis (Skolnik et al., Cell, 65:83-90, 1991) can be used.That is, a protein binding to the protein of the present invention canbe obtained by preparing a cDNA library from cells, tissues, organs (forexample, fetal liver, fetal kidney or cancer cell strain, and so on)expected to express a binding protein bound to the protein of thepresent invention by using a phage vector (λgt11, ZAP), expressing theprotein on LB-agarose, and fixing the protein expressed on the filter,reacting the purified and labeled protein of the present invention withthe above vector, and detecting a plaques expressing proteins bound tothe protein of the present invention by the label. As a method to labelthe protein of the invention is a method utilizing the binding betweenbiotin and avidin, or a method utilizing an antibody that specificallybinds to the protein of the present invention, or a peptide orpolypeptide (for example, GST) that is fused to the protein of thepresent invention, and a method using radioisotope or fluorescence andsuch.

Alternatively, as another embodiment of the method of screening of thepresent invention is a two-hybrid system utilizing cells (Fields et al.,Trends Genet., 10:286-292, 1994).

Proteins binding to the proteins of this invention or genes thereof canbe prepared utilizing the “two-hybrid system” (“MATCHMAKER Two-HybridSystem”, “Mammalian MATCHMAKER Two-Hybrid Assay Kit”, “MATCHMAKEROne-Hybrid System” (all from Clontech), “HybriZAP Two-Hybrid VectorSystem (Stratagene), (Dalton, S., and Treisman, R. (1992)Characterization of SAP-1, a protein recruited by serum response factorto the c-fos serum response element. Cell, 68:597-612), wherein theprotein of this invention is made to be expressed in yeast cells as aprotein fused to the DNA-binding domain of SRF or GAL4; a cDNA libraryis prepared from cells which are assumed to express proteins binding tothe protein of this invention so as to express the protein of thisinvention in the form fused to the VP16 or GAL4 transcriptionalactivation domain; the library is transferred to the aforementionedyeast cells to isolate the library-derived cDNA from the positive clonesdetected, which is then transferred to and expressed in E. coli (when aprotein binding to the protein of this invention is expressed in yeastcells, the reporter gene is activated due to the binding of bothproteins, enabling the identification of positive clones.)

As a reporter gene, for example, Ade2 gene, lacZ gene, CAT gene,luciferase gene, can be used besides HIS3 gene.

A protein binding to the protein of the present invention can bescreened by using an affinity chromatography. For example, the methodfor screening of the present invention utilizes affinity chromatography.The protein of the invention is immobilized on a carrier of an affinitycolumn, and a test sample, in which a protein capable of binding to theprotein of the invention is supposed to be expressed, is applied to thecolumn. A test sample herein may be, for example, cell extracts, celllysates, etc. After loading the test sample, the column is washed, andproteins bound to the protein of the invention can be prepared.

An amino acid sequence of the obtained protein is analyzed, an oligo DNAwas synthesized based on the sequence, and cDNA libraries are screenedusing the DNA as a probe to obtain a DNA encoding the protein.

A biosensor using the Surface Plasmon Resonance phenomenon may be usedas a mean for detecting or quantifying the bound compound in the presentinvention. When such a biosensor is used, the interaction between theprotein of the invention and a test compound can be observed inreal-time as a surface plasmon resonance signal, using only a minuteamount of proteins without labeling (for example, BIAcore, Pharmacia).Therefore, it is possible to evaluate the binding between the protein ofthe invention and a test compound using a biosensor such as BIAcore.

The method of screening molecules that bind when the immobilized proteinof the present invention is exposed to synthetic chemical compounds,natural substance banks, or a random phage peptide display library, orthe method of screening using high-throughput based on combinatorialchemistry techniques (Wrighton et al., “Small peptides as potentmimetics of the protein hormone erythropoietin”, Science, 273:458-64,1996; Verdine, “The combinatorial chemistry of nature”, Nature,384:11-13, 1996; Hogan, “Directed combinatorial chemistry”, Nature,384:17-19, 1996) is well known to one skilled in the art as a method forisolating not only proteins but also chemical compounds that bind toprotein of the present invention (including agonist and antagonist).

A compound isolated by the screening becomes a candidate for drugs whichpromote or inhibit the activity of the protein of the present invention,for treating or preventing of diseases attributed to, for example,functional abnormality of the protein of the present invention, orneoplasm. A substance obtained by converting, using addition, deletionand/or replacement, a part of the structure of a compound being obtainedusing the screening method of the present invention and having theactivity to bind to the protein of the present invention is alsoincluded in compounds obtained using the screening method of the presentinvention.

In addition, using the genes encoding the proteins of this invention orthe expression-controlling domains thereof, it is thought it possible toscreen compounds which can suppress or promote the expression (includingthe transcription and translation) of these genes in vivo. Thisscreening method can be utilized, for example, to screen candidatecompounds for therapeutic and preventive products for cancer.

This screening can be performed by a method comprising the steps of: (a)contacting a test sample with cells expressing the genes of thisinvention, (b) detecting the expression of the genes of this inventionin the cells, and (c) selecting a compound that decreases or increasesthe expression of the genes of this invention compared with that in thecase where the test sample is not contacted with the cells (control).

Desired compounds can be screened, for example, by culturing a suitablecell strain which expresses the “fls353” or “fls485” gene (such as HeLaS3, Hep G2, etc.) together with a test sample and investigating theexpression of these genes (including transcription and translationthereof) by detecting mRNA using northern blot technique or RT-PCRmethod, by detecting protein using western blotting, or these methodsmodified, and selecting compounds to increase or decrease the expressionof these genes compared with the case where no test sample is added.

It is also possible to screen compounds to suppress or promote the geneexpression of this invention in vivo by a method employing theactivation or inactivation of the expression-controlling domain for thegene of this invention as an indicator. This screening can beimplemented by a method comprising the steps of (a) providing cells intowhich a vector comprising a reporter gene functionally linked downstreamof the expression control region of the gene of this invention, (b)contacting a test sample with the cells, (c) detecting the activity ofthe reporter gene in the cells, and (d) selecting a compound thatdecreases or increases the activity compared with that in the case wherethe test sample is not contacted with the cells (control).

Herein, “functionally linked” means that the expression-controllingdomain and a reporter gene are linked in such a way that the reportergene linked to downstream of the expression-controlling domain can beexpressed in response to the activation of the expression-controllingdomain.

For example, after cloning the expression-controlling domain (promoter,enhancer, etc.) of the gene of this invention by screening a genomic DNAlibrary with the nucleotide sequence set forth in SEQ ID NOs:1, 3 or 5or portions thereof as the probe an expression vector is prepared toinsert the domain to upstream of an appropriate reporter gene(chloramphenicol acetyltransferase gene, luciferase gene, etc.), andtransferred to a mammalian cell line. Next, by contacting test sampleswith the cell line to detect the reporter activity, and selecting acompound to increase or decrease the reporter activity compared withthat in the cells contacted with no test samples, compounds to suppressor promote the expression of the gene of this invention can be screened.Detecting the expression of the gene of this invention with the reporteractivity as an index, this screening method is characteristic in itssimplicity and easiness compared with the above-described directdetection method such as northern analysis.

These compounds that are isolated by screening and that promote orsuppress the expression of the gene of this invention can be candidatesof medicines for various disorders caused by the aberrant expression ofthe gene, and expected to be used as drugs to prevent or treat disorderssuch as cancer in particular. In addition, substances obtainable byusing the above-described screening method, and having the structures ofcompounds to promote or suppress the gene expression of this inventionwhich are partially modified by addition, deletion and/or substitutionare also included in the compounds obtained by using the screeningmethod of this invention.

When the compound obtained by the screening method of the invention isused as a pharmaceutical for humans and other mammals, such as, mice,rats, guinea pigs, rabbits, chicken, cats, dogs, sheep, pigs, bovines,monkeys, baboons, chimpanzees, the isolated compound can be administerednot only directly, but also a dosage form using known pharmaceuticalpreparation methods. For example, according to the need, the drugs canbe taken orally as sugarcoated tablets, capsules, elixirs andmicrocapsules or non-orally in the form of injections of sterilesolutions or suspensions with water or any other pharmaceuticallyacceptable liquid. For example, the compounds can be mixed withpharmacologically acceptable carriers or medium, specifically,sterilized water, physiological saline, plant-oil, emulsifiers,suspending agent, surface-active agent, stabilizers, flavoring agents,excipients, vehicles, preservatives and binders, into a unit dose formrequired for generally accepted drug implementation. The amount ofactive ingredients in these preparations makes a suitable dosage withinthe indicated range acquirable.

Examples for additives which can be mixed to tablets and capsules are,binders such as gelatin, corn starch, tragacanth gum and gum acacia;excipients such as crystalline cellulose; swelling agents such as cornstarch, gelatin and alginic acid; lubricants such as magnesium stearate;sweeteners such as sucrose, lactose or saccharin; flavoring agents suchas peppermint, Gaultheria adenothrix oil and cherry. When the unitdosage form is a capsule, a liquid carrier such as oil can also beincluded in the above ingredients. Sterile composites for injections canbe formulated following normal drug implementations using vehicles suchas distilled water used for injections.

Physiological saline, glucose, and other isotonic liquids includingadjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodiumchloride, can be used as aqueous solutions for injections. These can beused in conjunction with suitable solubilizers, such as alcohol,specifically ethanol, polyalcohols such as propylene glycol andpolyethylene glycol, and non-ionic surfactants such as Polysorbate 80(TM) and HCO-50.

Sesame oil or Soy-bean oil can be used as a oleaginous liquid and may beused in conjunction with benzyl benzoate or benzyl alcohol as asolubilizers; may be formulated with a buffer such as phosphate bufferand sodium acetate buffer, a pain-killer such as procaine hydrochloride,a stabilizer such as benzyl alcohol and phenol, and an anti-oxidant. Theprepared injection is filled into a suitable ampule.

Methods well known to one skilled in the art may be used to administer apharmaceutical compound to patients, for example as intraarterial,intravenous, percutaneous injections and also as intranasal,transbronchial, intramuscular or oral administrations. The dosage variesaccording to the body-weight and age of a patient and the administrationmethod, but one skilled in the art can suitably select them. If thecompound can be encoded by a DNA, the DNA can be inserted into a vectorfor gene therapy and perform the therapy. The dosage and method ofadministration vary according to the body-weight, age, and symptoms of apatient, but one skilled in the art can select them suitably.

For example, although there are some differences according to thesymptoms, the dose of a compound that binds with the transcriptionalregulatory factor of the present invention and inhibits its activity isabout 0.1 mg to about 100 mg per day, preferably about 1.0 mg to about50 mg per day and more preferably about 1.0 mg to about 20 mg per day,when administered orally to a normal adult (weight 60 kg).

When administering parenterally in the form of an injection to a normaladult (weight 60 kg), although there are some differences according tothe patient, target organ, symptoms and method of administration, it isconvenient to intravenously inject a dose of about 0.01 mg to about 30mg per day, preferably about 0.1 to about 20 mg per day and morepreferably about 0.1 to about 10 mg per day. Also, in the case of otheranimals too, it is possible to administer an amount converted to 60 kgsof body-weight.

All references and patents cited herein are incorporated by reference intheir entirety.

EXAMPLES

In the following, the present invention will be more specificallydescribed with reference to examples, but is not be construed as beinglimited thereto.

Example 1 Preparation of Subtractive Library of Fetal Gene

By the suppression subtractive hybridization method with cDNAs derivedfrom fetal livers as the tester and cDNAs derived from adult livers asthe driver, the subtraction method was performed to prepare asubtractive library comprising fetal genes expressed specifically ormore prominently in the fetal liver.

A subtraction library was prepared using the PCR-Select™ cDNASubtraction kit (CLONTECH) basically according to the method describedby Luda Diatchenko et al. (Proc. Natl. Acad. Sci. USA, 93:6025-6030,1996).

First, double-stranded cDNAs were synthesized from polyA⁺ RNA preparedfrom human fetal (CLONTECH) and adult livers (CLONTECH) by the standardmethod using MMLV reverse transcriptase. Next, the respective cDNAs wereblunt-ended with T4 DNA polymerase, and then cleaved by RsaI. A part ofthe cDNA originating from fetal liver (tester) was split in two; one ofwhich was ligated with the adapter-1 and the other with the adapter-2.Each aliquot was mixed with 120-fold amount of the adult liver cDNA(driver), denatured by heat, and subjected to the first hybridization at68° C. for 8 hours. Aliquots were then combined without heatdenaturation, mixed further with an excess amount of heat-denatureddriver, and subjected to the second hybridization at 68° C. for about 16hours. The mixture was diluted with the dilution buffer, incubated at75° C. for 7 minutes to remove the shorter strands of adapters, and usedas a template for PCR. By performing PCR with primers corresponding tothe adapters, PCR primers 1 and 2, cDNAs carrying different adapters ontheir two ends (subtracted cDNAs) were selectively amplified(suppression PCR). To obtain products with further selectivity, aportion of the amplified products was used as a template for PCR withprimers Nested PCR primers 1 and 2, which locate further inside of theprimers; PCR primers 1 and 2. The products were purified using the“QIAquick PCR Purification kit” (QIAGEN), and cloned into the pT7Blue-Tvector (Novagen) by the TA cloning method to create a subtractionlibrary.

Adaptors 1 and 2, primers 1 and 2, nested PCR primers 1 and 2 are shownin Table 1.

TABLE 1 Adaptor 1 5′ CTAATACGACTCACTATAGGGC (SEQ ID NO: 7)TCGAGCGGCCGCCCGGGCAGGT 3′ 3′ GGCCCGTCCA 5′ (SEQ ID NO: 8) Adaptor 2 5′TGTAGCGTGAAGACGACAGAAA (SEQ ID NO: 9) GGGCGTGGTGCGGAGGGCGGT 3′ 3′GCCTCCCGCCA 5′ (SEQ ID NO: 10) PCR primer 1 5′ CTAATACGACTCACTATAGGGC 3′(SEQ ID NO: 11) PCR primer 2 5′ TGTAGCGTGAAGACGACAGAA 3′ (SEQ ID NO: 12)Nested PCR primer 1 5′ TCGAGCGGCCGCCCGGGCAGGT 3′ (SEQ ID NO: 13)Nested PCR primer 2 5′ AGGGCGTGGTGCGGAGGGCGGT 3′ (SEQ ID NO: 14)

Example 2 Construction of Fetal Gene EST

By sequencing clones extracted from the library prepared in Example 1 atrandom, EST comprising fetal genes was constructed.

Plasmid DNA prepared by the alkali SDS method or products of colony PCRwere used as a template for sequence reaction. Sequence reaction wasperformed by the cycle-sequencing method using the ABI PRISM™ DyeTerminator Cycle Sequencing Ready Reaction Kit With AmplyTaq DNAPolymerase, FS (Perkin Elmer), and the result was analyzed by the ABI377 DNA Sequencer.

Colony PCR was performed as follows. Colonies carrying recombinantvectors were directly suspended into PCR reaction mixtures that containvector primers, “M13 P4-22 primer” (5′ CCAGGGTTTTCCCAGTCACGAC 3′: SEQ IDNO:15) and “M13 P5-22 primer” (5′ TCACACAGGAAACAGCTATGAC 3′: SEQ IDNO:16). After PCR reaction, amplified insert DNA was separated fromunreacted primers and nucleotides and such by gel filtration or thelike, and used as a template for sequencing.

Example 3 RT-PCR Assay

As a result of EST (expressed sequence tags) construction by sequencing,primer sets specific for 84 kinds of ESTs having novel sequences wereprepared respectively, and screening was performed to examine theexpression of each gene in fetal liver, kidney, lung and brain as wellas in adult liver, kidney, lung, brain, bone marrow and testis byRT-PCR.

Single-stranded cDNAs were synthesized from polyA⁺ RNA (CLONTECH) or thetotal RNA by the standard method using a SUPERSCRIPT™ II RNase H⁻Reverse Transcriptase (GIBCO BRL), and a portion of the cDNAs thusobtained was used as the template for RT-PCR assay. PCR was carried outprincipally under the following conditions.

Composition of the Reaction Solution

-   -   TakaRa Taq (TaKaRa) 0.24 μl    -   TaqStart™ Antibody (CLONTECH) 0.24 μl    -   10×PCR buffer (TaKaRa) 3 μl    -   2.5 mM dNTPs (TaKaRa) 2.4 μl    -   20 μM primer 0.6 μl each    -   Template DNA 6 μl    -   /total volume 30 μl

Reaction Conditions

1 cycle of 94° C. for 2 min; 28 to 35 cycles of 94° C. for 30 s, 50° C.to 60° C. for 1 min, and 72° C. for 1 min; and 1 cycle of 72° C. for 3min.

Sixty-three clones that showed expression patterns specific in fetaltissues were selected, and then examined for their expressions inclinical samples of colon cancer and a variety of cancer cell strains.As a result, clones “fls353” and “fls485” were selected as the fetalgenes with the expression activated in cancer cells.

Primer sets used in RT-PCR for amplifying “fls353” and “fls485”, andG3PDH used as the control are shown in Table 2.

TABLE 2 fls353 FLS353 S1 primer 5′ GATGCTTTCGTA (SEQ ID NO: 17)GTTCAGATGTAA 3′ FLS353 A1 primer 5′ AATGCAGCAAGA (SEQ ID NO: 18)GGTGGTGGAGAT 3′ fls485 FLS485 S1 primer 5′ GAGGGGGATCAG (SEQ ID NO: 19)CTCAATGGTCTG 3′ FLS485 A1 primer 5′ ACCCCATCGTGG (SEQ ID NO: 20)ACTTCCCTCTGC 3′ G3PDH HG3 S1 primer 5′ TCATCATCTCTG (SEQ ID NO: 21)CCCCCTCTGCTG 3′ HG3 A1 primer 5′ GACGCCTGCTTC (SEQ ID NO: 22)ACCACCTTCTTG 3′

Example 4 Cloning of Full Length cDNA of fls353

A 5′-stretch plus cDNA library derived from human fetal liver (CLONTECH)was screened by the plaque hybridization method with the fls353 originalclone as the probe. Screening of about 5×10⁵ plaques resulted in gaininga few positive clones. DNA inserts of these positive clones wereamplified by PCR and directly sequenced to determine their sequences.Public database search using the sequences thus obtained several ESTswhich coincided with the 3′-terminus of fls353. Based on these ESTsequences, FLS353 E2 primer (5′-CTGGTGCAGTTGGTGAGGTTTTCT 3′/SEQ IDNO:23) and FLS353 R1 primer (5′-CAATCACCGTCCCCAAGTCACCAG 3′/SEQ IDNO:24) were prepared, and the 3′-terminal fragments of fls353 gene wereamplified by PCR with cDNA derived from human fetal liver as thetemplate. Amplified fragments were subcloned into the pT7Blue-T vector(Novagen), and the nucleotide sequence of the 3′-terminal fragmentcontaining a stop codon for fls353 gene was determined by sequencing aplurality of subclones. In addition, the 5′-terminus of fls353 gene wascloned by the 5′ RACE (Rapid Amplification of cDNA Ends) method. Thatis, a 5′-terminal fragment (approximately 1.3 kbp) was amplified by PCRwith a Marathon™ Ready cDNA (CLONTECH) as the template using the FLS353S1 primer and AP1 primer (CLONTECH), and PCR products were subclonedinto the pT7Blue-T vector. A plurality of subclones were sequenced todetermine the nucleotide sequence of the 5′-terminus of fls353 genecontaining the initiation codon.

cDNA containing the entire coding region was amplified from the fetalliver-derived cDNAs and such by PCR using FLS353 WN1 primer(5′-AGTCGCGGCCGCCGGTATGCAGAGAAGAGGACAGAA-3′/SEQ ID NO:25) and WN2 primer(5′-AGTCGCGGCCGCAAAAGGGGTGAAAGAGAAGATTGC-3′/SEQ ID NO:26).

Example 5 Cloning of Full-Length cDNA of fls485

Human testis cDNA library (CLONTECH) was plated on the plates by thestandard method, and then transferred onto nylon membrane (Hybond N+).This membrane was cut into fragments presumed to contain about 200plaques each, and the phage on the membrane was extracted into a lambdadilution buffer (SM). With the cDNA pool thus obtained as a template,screening was performed by PCR using the above-described FLS485 S1primer and FLS485 A1 primer to identify a pool containing positiveclones. With this pool as a template, a fragment (about 800 bp) of thispositive clone was amplified using the above-described FLS485 A1 primerand GT10 S1 primer (5′-CTTTTGAGCAAGTTCAGCCT-3′/SEQ ID NO:27) which is avector-primer of the library (λgt10 vector). After the sequence wasconfirmed, using this fragment as a probe, a human fetal liver-derived5′-stretch plus cDNA library (CLONTECH) was screened by the plaquehybridization method. Screening of about 10⁶ plaques yielded 8 positiveclones. DNA inserts of these positive clones were amplified by PCR anddirectly sequenced. Based on the sequence thus obtained, FLS485 A4primer (5′-TTGAAATGTCCACTCGCTTATCCT-3′/SEQ ID NO:28) was prepared, andthe 5′-terminal fragment was cloned by the 5′ RACE (Rapid Amplificationof cDNA Ends) method. A 5′-terminal fragment (approximately 600 bp) wasamplified by PCR with a human fetal liver-derived Marathon™ Ready cDNA(CLONTECH) as a template using FLS485 A4 primer and AP1 primer(CLONTECH), and PCR-amplified products were subcloned into the pT7Blue-Tvector (Novagen). A plurality of subclones was sequenced to determinethe nucleotide sequence of the 5′-terminus of fls485 gene containing theinitiation codon.

cDNA containing the entire coding region was amplified and cloned fromthe human fetal liver-derived cDNAs and such by PCR using a combinationof the 5′-terminal sense primer and 3′-terminal antisense primerdescribed in Table 3.

TABLE 3 5′-terminal sense primer S1 primer 5′ AGTCGCGGCCGCGCTAAG(SEQ ID NO: 29) CAGGTGCGGAGGGGAGTC 3′ S2 primer 5′ AGTCGCGGCCGCCAGATA(SEQ ID NO: 30) TTCTTCCCACCTTTGGAG 3′ 3′-terminal antisense primerA1 primer 5′ AGTCGCGGCCGCGAGGAG (SEQ ID NO: 31) CTGTATAAGGGGTTGGAG 3′A2 primer 5′ AGTCGCGGCCGCTGCCAG (SEQ ID NO: 32) GGTTTGTATGTGATTGTC 3′

Example 6 Analyses of fls353

6-1 Expression Distribution in Normal Tissues

Expression distribution of fls353 in normal tissues was examined bynorthern analysis. The original clone of fls353 was labeled with[α-³²P]dCTP by the random primer method using the Ready-to Go DNAlabeling beads (Pharmacia) to be used as a probe. Hybridization wasperformed at 68° C. in the ExpressHyb Hybridization Solution (CLONTECH)according to the method recommended by the manufacturer using theMultiple Tissue Northern (MTN) Blot—Human, Human II, Human III, HumanFetal II, Human Cancer Cell Line (CLONTECH). Final washing was performedin 0.1×SSC and 0.1% SDS at 50° C.

The results are shown in FIG. 1. Prominent expression of fls353 wasdetected in fetal liver, kidney, lung and brain, but almost noexpression of it was seen in the corresponding adult tissues. Amongother adult tissues, relatively strong expression was observed in thymusand testis, and only very weak expression was seen in other tissues.

6-2 Expression in Cancer Cell Strains

First, analyses of the fls353 expression in cancer cell strainssimilarly performed by the aforementioned northern hybridizationdetected its strong expression in 7 out of 8 cancer cell strainsexamined (FIG. 1). Subsequent examination of the fls353 expression inclinical samples of colon cancer by RT-PCR (FIGS. 2 and 3) resulted inthe detection of its expression in 10 different types of tumors out of12 types of samples. As the controls for 6 types out of these tumors,the fls353 expression was simultaneously examined in normal tissuestaken from the same patient, but practically no expression was detected,indicating that the activation of fls353 expression is induced bymalignant transformation of colon tissue cells.

6-3 Expression of fls353 in Clinical Cancer Samples

Northern analysis of fls353 expression in clinical samples of hepatomaand lung cancer (FIGS. 10 and 11) resulted in a clear demonstration ofits activated expression in cancer tissues. These results indicate thatthe fls353 expression is activated in a variety of cancer types, andinvolved in the progress of these cancers.

6-4 Structural Analyses

cDNA covering the entire coding sequence of the fls353 gene was clonedby screening the cDNA library using the plaque hybridization method andRACE method and such, and sequenced. As a result, fls353 was proved toencode a protein comprising 747 amino acid residues (FIGS. 4 and 5).Nucleotide sequence of the fls353 cDNA thus determined and amino acidsequence encoded by the cDNA are shown in SEQ ID NO:1 and 2,respectively. Database search using this amino acid sequence detected noprotein showing a significant homology to the sequence. The fls353protein is structurally characteristic in that it has the ATP/GTPbinding consensus sequence (aa52-59,[Ala/Gly]-Xaa-Xaa-Xaa-Xaa-Gly-Lys-[Ser/Thr] (SEQ ID NO:35), where Xaarepresents any amino acid residue), and that its content percentage ofbasic amino acid, Lys, is high (13.2%), and assumed to be a basicprotein (calculated isoelectric point pI=9.62). However, no motifpredicting the function of this protein was detected.

Example 7 Analyses of fls485

7-1 Expression Distribution in Normal Tissues

Expression of fls485 in normal tissues was examined by northern analysis(FIG. 6), which was performed similarly as the above-described fls353using the original clones of fls485 as a probe.

As a result, it has been proved that fls485 is a gene which is expressedwith an extremely high selectivity in organs such as fetal liver andkidney as well as adult testis and small intestine containing manyundifferentiated cells, wherein cell differentiation and proliferationare actively in progress.

7-2 Expression in Tumor Cell Strains

Examination of fls485 expression in cultured cells by RT-PCT analysishas revealed that fls485 exists in several hepatoma-derived cellstrains—HepG2, HuH7 and PLC/PRF5 (Alexander), though not detected inprimary culture of lung fibroblast which are normal cells (FIG. 7).Expression pattern of fls485 in hepatoma-derived cell strains coincidedvery well with that of a fetoprotein, a hepatoma tumor marker,indicating that a close correlation of fls485 expression with cellstrains more reminiscent of the property of cancer cells in adulttissues. No expression of fls485 was detected in the adult liver at allon the analytical level either by northern analysis or even by the moresensitive RT-PCR analysis. These results indicate that fls485 expressionis activated accompanied with a progress of malignant transformation ofhepatocytes.

Also, the database search detected registration of four ESTs predictedto be derived from fls485 in a public database. Three out of these ESTsare derived from colon cancer cell strains (AA308814 and AA305159 arederived from the large intestine adenoma strain Caco2; AA622964 isderived from the colon cancer strain RER+). These results indicate thefls485 expression is also activated in certain types of colon cancers.

7-3 Expression in Clinical Cancer Samples

Northern analysis of fls485 expression in clinical samples of hepatomaand lung cancer (FIGS. 10 and 11) resulted in a clear demonstration ofits activated expression in cancer tissues. These results indicate thatthe fls353 gene expression is activated in a variety of cancer types,and involved in the progress of these cancers.

7-4 Structural Analyses

Cloning of cDNA covering the entire coding region of fls485 has provedthat the gene encodes a protein comprising 353 amino acid residues (FIG.8). Nucleotide sequence of the full-length fls485 cDNA thus determinedand the amino acid sequence encoded by the cDNA are shown in SEQ IDNOs:3 and 4, respectively. This amino acid sequence is characteristic asfollows:

1. In the central portion, there exists a sequence predicted to be azinc finger domain like C4-type. This domain is in the form of tworepeats of a structural unit comprising two consecutive sequences eachcomprising Cys-Xaa-Xaa-Cys-Xaa-Gly-Xaa-Gly (SEQ ID NO:33) followed byone sequence comprising Cys-Xaa-Xaa-Cys-Xaa-Gly (SEQ ID NO:34).

2. The C-terminal sequence fits the consensus sequence of aCys-Aaa-Aaa-Xaa (SEQ ID NO:36) box (Aaa mainly represents aliphaticamino acid). This motif is often observed in G-protein including rasproteins, and supposed to be a necessary motif for the mechanism tolocalize Cys to the inner side of cell membrane through its modificationwith lipids. No other signal for the intracellular localization isdetected.

3. Furthermore, the C-terminal sequence also fits the (Ser/Thr)-Xaa-Valmotif which is supposed to bind to a characteristic domain structurereferred to as PDZ (DHR) domain which has been recently elucidated. Thisbinding motif is observed in the binding of APC, a causative gene offamilial polyposis, and human homologue of tumor suppressor gene DLG ofDrosophila, which has been recognized as an important and principalbinding motif in the intracellular signal transduction.

4. This protein is hydrophilic on the whole, and transmembrane domain,secretory signal, and such are not detected therein.

Homology search detected no protein having a significant homology tofls485 in database, although homology to several proteins was observedin the zinc finger-like domain.

Although the zinc finger domain is a structural domain often found in alarge number of transcriptional regulatory factors, a zinc finger-likedomain of fls485 is different from those of such a typicaltranscriptional regulatory factor type, and, furthermore, no sequencethought to be a nuclear transport signal has been detected in the fls485amino acid sequence.

In addition, analysis by PCR has demonstrated the presence of two typesof mRNAs for fls485, one comprising a sequence of 146 bp present in the5′-terminus (fls485 L/SEQ ID NO:3) and the other skipping the sequence(fls485 S/SEQ ID NO:5) (FIG. 9). Since this region, underlined in FIG.8, corresponds to the N-terminus of the putative coding region, fls485 Lis able to encode a protein longer by 51 amino acids than fls485 S(amino acid sequence of fls485 L protein is shown in SEQ ID NO:4, andthat of fls485 S protein in SEQ ID NO:6). Furthermore, northern analysisdemonstrated the presence of a longer transcript in testis than thosefound in other tissues. However, analysis by PCR proved that thistranscript has the same coding region as that in other tissues, and thiswas thought to be due to the difference in length in the 3′-untranslatedregion (UTR) (possibly due to the difference in the polyA additionsite).

Industrial Applicability

The present invention has provided novel proteins which are specificallyor more prominently expressed in fetal tissues, and genes encoding theproteins. The genes are strongly expressed in tumor cells, and assumedto participate in malignant transformation of cells. Therefore,expression inhibitors for fetal genes of this invention are expected tobe useful as antitumor agents. Furthermore, it is possible to utilizethe fetal genes of this invention and proteins encoded by the genes asthe tool for purification and cloning of novel factors involved in cellproliferation, immortalization, cellular infiltration, metastasis andangiogenesis, and further as the tool for the development of drugs forvarious disorders caused by the aberrant activation of gene expressionof this invention due to the irregularity of expression control in vivo.It is thought it possible to develop medicines depending on novel actionmechanisms by designing drugs targeted on the genes of this invention.

1. An isolated polypeptide comprising the sequence of SEQ ID NO:
 4. 2.The isolated polypeptide of claim 1, wherein the amino acid sequence ofthe polypeptide consists of SEQ ID NO:
 4. 3. The isolated polypeptide ofclaim 1, wherein the polypeptide is substantially pure.
 4. The isolatedpolypeptide of claim 3, wherein the polypeptide is at least 95% pure bydry weight.
 5. The isolated polypeptide of claim 3, wherein thepolypeptide is at least 75% pure by dry weight.
 6. An isolatedimmunogenic fragment of the polypeptide of amino acid sequence SEQ IDNO: 4, wherein the immunogenic fragment is at least 7 amino acids inlength.
 7. The isolated immunogenic fragment of claim 6, wherein theimmunogenic fragment is 8 amino acids or more in length.
 8. The isolatedimmunogenic fragment of claim 7, wherein the immunogenic fragment is 9amino acids or more in length.
 9. The isolated immunogenic fragment ofclaim 6, wherein the immunogenic fragment is at least 10% of the lengthof the polypeptide of amino acid sequence SEQ ID NO:
 4. 10. The isolatedimmunogenic fragment of claim 9, wherein the immunogenic fragment is atleast 20% of the length of the polypeptide of amino acid sequence SEQ IDNO:
 4. 11. The isolated immunogenic fragment of claim 10, wherein theimmunogenic fragment is at least 40% of the length of the polypeptide ofamino acid sequence SEQ ID NO:
 4. 12. The isolated immunogenic fragmentof claim 11, wherein the immunogenic fragment is at least 60% of thelength of the polypeptide of amino acid sequence SEQ ID NO:
 4. 13. Amethod for preparing an antibody, the method comprising immunizing ananimal with the isolated polypeptide of claim 1 and obtaining anantibody that specifically binds to the polypeptide.
 14. The method ofclaim 13, wherein the amino acid sequence of the isolated polypeptideconsists of SEQ ID NO:
 4. 15. The method of claim 13, wherein theantibody is a polyclonal antibody, IgG, or IgM.
 16. The method of claim13, wherein the antibody is a human antibody.
 17. The method of claim13, further comprising generating a hybridoma from a cell of the animal,wherein the antibody that specifically binds to the polypeptide is amonoclonal antibody produced by the hybridoma.
 18. The method of claim17, further comprising generating a DNA encoding a single chain Fv(scFv) form of the monoclonal antibody.
 19. A method for screening for atest sample that binds the isolated polypeptide of claim 1, the methodcomprising: (a) contacting a test sample with the isolated polypeptideof claim 1; (b) detecting whether the test sample binds to the isolatedpolypeptide of claim 1; and (c) selecting the test sample if it binds tothe isolated polypeptide of claim
 1. 20. The method of claim 19, whereinthe amino acid sequence of the isolated polypeptide consists of SEQ IDNO:
 4. 21. The method of claim 19, wherein the test sample comprises aprotein.
 22. The method of claim 19, wherein the test sample comprises asynthetic chemical compound.
 23. An isolated polypeptide comprising thesequence of SEQ ID NO:
 6. 24. The isolated polypeptide of claim 23,wherein the amino acid sequence of the polypeptide consists of SEQ IDNO:
 6. 25. The isolated polypeptide of claim 23, wherein the polypeptideis substantially pure.
 26. The isolated polypeptide of claim 25, whereinthe polypeptide is at least 95% pure by dry weight.
 27. The isolatedpolypeptide of claim 25, wherein the polypeptide is at least 75% pure bydry weight.
 28. An isolated immunogenic fragment of the polypeptide ofamino acid sequence SEQ ID NO: 6, wherein the immunogenic fragment is atleast 7 amino acids in length.
 29. The isolated immunogenic fragment ofclaim 28, wherein the immunogenic fragment is 8 amino acids or more inlength.
 30. The isolated immunogenic fragment of claim 29, wherein theimmunogenic fragment is 9 amino acids or more in length.
 31. Theisolated immunogenic fragment of claim 28, wherein the immunogenicfragment is at least 10% of the length of the polypeptide of amino acidsequence SEQ ID NO:
 6. 32. The isolated immunogenic fragment of claim31, wherein the immunogenic fragment is at least 20% of the length ofthe polypeptide of amino acid sequence SEQ ID NO:
 6. 33. The isolatedimmunogenic fragment of claim 32, wherein the immunogenic fragment is atleast 40% of the length of the polypeptide of amino acid sequence SEQ IDNO:
 6. 34. The isolated immunogenic fragment claim 33, wherein theimmunogenic fragment is at least 60% of the length of the polypeptide ofamino acid sequence SEQ ID NO:
 6. 35. A method for preparing anantibody, the method comprising immunizing an animal with the isolatedpolypeptide of claim 23 and obtaining an antibody that specificallybinds to the polypeptide.
 36. The method of claim 35, wherein the aminoacid sequence of the isolated polypeptide consists of SEQ ID NO:
 6. 37.The method of claim 35, wherein the antibody is a polyclonal antibody,IgG, or IgM.
 38. The method of claim 35, wherein the antibody is a humanantibody.
 39. The method of claim 35, further comprising generating ahybridoma from a cell of the animal, wherein the antibody thatspecifically binds to the polypeptide is a monoclonal antibody producedby the hybridoma.
 40. The method of claim 39, further comprisinggenerating a DNA encoding a single chain Fv (scFv) form of themonoclonal antibody.
 41. A method for screening for a test sample thatbinds the isolated polypeptide of claim 23, the method comprising: (a)contacting a test sample with the isolated polypeptide of claim 23; (b)detecting whether the test sample binds to the isolated polypeptide ofclaim 22; and (c) selecting the test sample if it binds to the isolatedpolypeptide of claim
 22. 42. The method of claim 41, wherein the aminoacid sequence of the isolated polypeptide consists of SEQ ID NO:
 6. 43.The method of claim 41, wherein the test sample comprises a protein. 44.The method of claim 41, wherein the test sample comprises a syntheticchemical compound.